SUMMARY/ABSTRACT (PROJECT 2) Immunotherapy has revolutionized the treatment of a variety of advanced malignancies. Anti-CD19 chimeric antigen receptor redirected T cells (CART-19) have been particularly successful in B-cell malignancies. How to translate the success of CART cell therapy to other malignancies such as acute myeloid leukemia (AML) remains an important question in the field. A critical requirement of CART cell therapy is that the target tissue be expendable. AML is a malignancy of the hematopoietic stem/progenitor cells (HSPC) and shares cell surface antigens with normal HSPC and with normal myeloid progeny such as neutrophils and monocytes. It has become clear that the lack of AML-specific antigens is the single biggest impediment to unleashing the power of CART cells against AML and other myeloid malignancies. The long-term goal of this project is to develop a clinically feasible CART cell platform for AML, by creating AML-specific CART cells that are able to expand and persist in vivo to eradicate AML while preserving normal marrow function. The central hypothesis is that a durable anti-leukemic effect from anti-CD33 CART cells can co-exist with adequate levels of genetically engineered CD33-deficient hematopoiesis. This will be accomplished in three specific aims. In Aim 1, high quality depletable anti-CD33 CAR T cells will be manufactured. These will be allogeneic, donor-derived T cells transduced with a biscistronic lentiviral vector that encodes a humanized anti-CD33-41BB-zeta CAR as well as CD20. In Aim 2, the feasibility and safety of manufacturing CD33-deficient human HSPC will be tested and regulatory approvals to manufacture CD33-deficient HSPC will be obtained. In Aim 3, a clinical trial will be conducted of a combined approach incorporating CD33-deficient, CAR-resistant allogeneic HCT followed by CART-33 infusion in patients with AML. This research will be significant because it will contribute depth (of clinical responses) and breadth (of eligibility for potentially curative therapy) to the therapeutic arsenal against AML. The innovation of the proposed research lies in replacing the search for suitable leukemia-specific antigens with a novel platform that combines pan-myeloid specific CART (such as CART-33) with an infusion of donor HSPC that are genetically engineered to lack CD33 and which are therefore resistant to killing by CART-33.